Abstract

In plant organs and tissues, the neutral storage lipids are confined to discrete spherical organelles called oil bodies. Oil bodies from plant seeds contain 0.6–3% proteins, including oleosins, steroleosins, and caleosins. In this study, a caleosin isoform of ~30kDa was identified in the olive pollen grain. The protein was mainly located at the boundaries of the oil bodies in the cytoplasm of the pollen grain and the pollen tube. In addition, caleosins were also visualized in the cytoplasm at the subapical zone, as well as in the tonoplast of vacuoles present in the pollen tube cytoplasm. The cellular behaviour of lipid bodies in the olive pollen was also monitored during in vitro germination. The number of oil bodies decreased 20-fold in the pollen grain during germination, whereas the opposite tendency occurred in the pollen tube, suggesting that oil bodies moved from one to the other. The data suggest that this pollen caleosin might have a role in the mobilization of oil bodies as well as in the reorganization of membrane compartments during pollen in vitro germination.

Introduction

Eukaryotes contain large amounts of neutral lipids (Murphy, 2001). In plant organs and tissues (e.g. seeds), the neutral storage lipids are confined to discrete spherical organelles of ~0.5–2μm (Huang, 1996) called oil bodies (OBs). They have been proposed to consist of a core containing neutral lipids [triacylglycerols (TAGs), sterol esters, etc.] surrounded by a single layer of phospholipids with a few embedded unique proteins (Tzen et al., 1993; Huang, 1996). OBs represent the primary energy reserve to support periods of active metabolism (Murphy and Vance, 1999). However, despite OBs having often been regarded as simple storage sites, a plethora of unsuspected dynamic roles has been suggested for these organelles, including subcellular lipid trafficking and turnover, and calcium signalling (Frandsen et al., 2001; Poxleitner et al., 2006).

Olive (Olea europaea L.) is a typical oil-storing plant species. Several olive organs and tissues have been reported to contain large amounts of OBs (Pacini and Juniper, 1979; Ross et al., 1993; Alché et al., 1999a, 2006; Rodríguez-García et al., 2003). The olive pollen grain accumulates a high number of OBs at maturity, which spread over the cytoplasm of the vegetative cell. During pollen hydration, these organelles polarize near the aperture through which the pollen tube emerges (Rodríguez-García et al., 2003). This fact suggests that OBs might be involved in olive pollen germination and early pollen tube growth in the pistil. However, nothing is known about the behaviour of OBs during such a process. Recently, a unique caleosin isoform distinct from that in seed OBs has been identified in lily pollen (Jiang et al., 2008). Nevertheless, no research data regarding the presence and function of caleosins during pollen germination and pollen tube growth have been published to date.

The present paper reports the identification of a new OB-associated caleosin in olive (O. europaea L.) pollen and its cellular location during in vitro germination. The behaviour of OBs during in vitro pollen germination was also studied. Finally, the possible involvement of this caleosin in the mobilization of OBs and the reorganization of membrane compartments during this process is discussed.

Materials and methods

Pollen material and sampling

Olive (O. europaea L. cv. Picual) mature pollen grains were harvested from dehiscent anthers by vigorous shaking of flowering shoots inside large paper bags. Sampling was carried out from discrete trees of the collection of olive varieties of the CIFA ‘Venta del Llano’ (Jaén, Spain). Pollen samples were sieved through an appropriate set of meshes to remove floral debris and processed fresh or stored at –80°C until use.

In vitro germination of olive pollen

Pollen was pre-hydrated by incubation in a humid chamber at room temperature for 30min and then transferred to Petri dishes (0.1g per dish) containing 10ml of germination medium [10% (w/v) sucrose, 0.03% (w/v) Ca(NO3)2, 0.01% (w/v) KNO3, 0.02% (w/v) MgSO4, and 0.01% (w/v) boric acid]. Petri dishes were maintained at room temperature in the dark, and pollen grains were sampled 1, 2, 3, 6, and 12h after the onset of the in vitro germination.

Purification of OBs and the microsomal fraction from olive pollen

The isolation of OBs and the microsomal fraction from olive pollen tubes was carried out as described by Hernández-Pinzón et al. (2001). All steps were performed at 4°C. Samples were ground in a homogenization buffer consisting of 100mM HEPES buffer (pH 7.5) containing 0.4M sucrose, 10mM KCl, 1mM EDTA, and 2mM dithiothreitol (DTT). Homogenates were centrifuged at 6000g for 2min to remove debris. They were then fractionated by centrifugation at 20000g for 20min. The lower supernatant was further centrifuged at 100000 g for 1h. The pellet, corresponding to the microsomal fraction, was recovered and then resuspended in homogenization buffer and stored at −20°C until use. The upper lipid pad was resuspended in 50mM TRIS-HCl buffer (pH 7.2) containing 1M NaCl, 9M urea, and 0.2% (w/v) Tween-20. The suspension was diluted with 0.5ml of homogenization buffer and layered with 4 vols of 0.1M sucrose in 100mM HEPES buffer (pH 7.5). OBs were recovered after centrifugation as above. Washing steps were performed according to Jiang et al. (2007). OBs were washed in an equal volume of 0.1% (v/v) Nonidet P40 (NP40) substitute (Sigma-Aldrich, St Louis, MO, USA) and 0.2M sucrose in 5mM sodium phosphate buffer, pH 7.5. They were then centrifuged at 15700g for 30min and the upper fraction was collected and mixed with an equal volume of 2M NaCl and 0.6M sucrose in 10mM sodium phosphate buffer (pH 7.5). Finally, OBs were recovered upon centrifugation as above, mixed with 0.6M sucrose in 10mM sodium phosphate buffer (pH 7.5), and stored at −20°C until use.

Staining of OBs

After in vitro germination, pollen samples were fixed in 4% (w/v) paraformaldehyde and 0.2% (v/v) glutaraldehyde in 0.2M sodium cacodylate buffer (pH 7.2) at 4°C overnight. After three washes in cacodylate buffer for 10min each, pollen samples were resuspended in an anti-fading solution of Citifluor (Sigma-Aldrich). Aliquots (50μl) of germinated pollen were mixed with 10μl of a solution of 0.1mg ml−1 Nile Red (Sigma-Aldrich) in acetone. Samples were observed with a C1 confocal laser scanning microscope (Nikon, Japan) using an argon (488nm) laser. Z-series images were collected and processed using the EZ-C1 Gold v.2.10 build 240 software (Nikon).

Aliquots (25μl) of purified pollen OBs were incubated either with a solution of Sudan Black B 70% (w/v) in ethanol at 60°C for 1min or with a solution of Nile Red as above. Samples were observed with an Axioplan epifluorescence microscope (Carl Zeiss, Germany). Digital images were recorded with a ProgRes C3 camera (Jenoptik Laser, Germany) using the ProgRes CapturePro software (Jenoptik Laser).

OB morphometry

Measurements of the number and size (the largest diameter) of OBs from pollen grains and pollen tubes after hydration and at different germination times (1, 3, 6, and 12h) were carried out from digital images recorded with a C1 confocal laser scanning microscope (Nikon) and processed using the software EZ-C1 Gold version 2.10 build 240 (Nikon). Z-series images of five germinated pollen grains from three independent experiments (n=15) were captured and used for measurements and statistical analysis. The mean and the standard deviation were calculated and plotted using the SigmaPlot software (Systat Software, Germany).

Analysis of OB dynamics in living pollen tubes

Pollen samples were germinated in vitro for 3h as described above. A 25μl aliquot of the germination medium was transferred onto a cover slide and mixed with 0.5μl of a solution containing 0.05mg ml−1 (w/v) Nile Red (Sigma-Aldrich) in 50% (v/v) acetone. Then, samples were immediately analysed with a C1 confocal laser scanning microscope (Nikon). Time-lapse capture was performed using the EZ-C1 Gold v.2.10 build 240 software (Nikon).

Protein extraction

Both mature and germinated pollen samples (0.1g) were resupended in 1.5ml of extraction buffer A [40mM TRIS-HCl, pH 7.4, 2% (v/v) Triton X-100, 1mg ml−1 ascorbic acid, 60mM DTT, 5% (w/v) polyvinylpyrrolidone (PVPP), and 15μl of a protease inhibitor cocktail (Sigma-Aldrich)] and stirred for 6h at 4°C. After centrifugation at 10000g for 30min at 4°C, the supernatants containing the eluted proteins were transferred to 15ml tubes and mixed with 10 vols of a solution consisting of 20% (w/v) trichloroacetic acid (TCA) and 0.2% (w/v) DTT in acetone. Proteins were precipitated at −20°C overnight. Samples were then centrifuged at 10000g for 30min at 4°C. The resulting pellets were rinsed with acetone three times for 15min each, resuspended in Laemmli (1970) sample buffer, dispensed into aliquots, and stored at −20°C.

The protein concentration in each sample was estimated by using the 2D Quant Kit (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer's instructions.

SDS–PAGE and western blotting

SDS–PAGE was performed according to Laemmli (1970). A 25μg aliquot of proteins per sample was loaded on 12% (w/v) polyacrylamide gels with 4% stacking gels using a Mini-Protean 3 apparatus (Bio-Rad). After electrophoresis, gels were stained with Coomassie Brilliant Blue R250 according to standard procedures. Proteins were electroblotted onto a PVDF membrane in a Semi-Dry Transfer Cell (Bio-Rad). The membrane was blocked overnight at 4°C in a solution containing 3% (w/v) bovine serum albumin (BSA) in TRIS-buffered saline (TBS) buffer, pH 7.4. Immunodetection of caleosins was carried out by incubation with a polyclonal antibody (Ab) prepared from the complete sequence of Clo3 caleosin from Arabidopsis thaliana, diluted 1:500 in TBS buffer (pH 7.4) containing 0.3% (v/v) Tween-20, for 12 h at 4°C. An Alexa Fluor 633-conjugated anti-rabbit IgG (Molecular Probes, Eugene, OR, USA), diluted 1:2000, served as the secondary Ab. The signal was detected in a Pharos FX molecular imager (Bio-Rad). The specificity of the anti-Clo3 Ab was tested by omitting this Ab from the protocol.

Caleosin immunoprecipitation

Total proteins were extracted from pollen and isolated OBs as described above. After TCA precipitation, pellets were resuspended in 200μl of a TBS solution containing 1mM phenylmethylsulphonyl fluoride (PMSF), 0.5% (v/v) NP40, 1% (v/v) Triton X-100, and 10μl (0.6μg) of the anti-Clo3 Ab. Samples were incubated for 4h at 4°C under gentle agitation, and 30μl of protein A–Sepharose were added. Samples were then incubated for 2h at 4°C under gentle agitation, and immunoprecipitated complexes were collected by centrifugation at 20000g for 15min at 4°C. The resulting pellets were washed three times with 0.5ml of TBS buffer containing 1% (v/v) Triton X-100 and 0.5% (v/v) NP40, and resuspended in 50μl of Laemmli sample buffer. SDS–PAGE and western blotting were conducted as described above. After electrophoresis, gels were stained with Coomassie Brilliant Blue R250 according to standard procedures. The specificity of the anti-Clo3 Ab was tested by omitting the primary Ab from the protocol.

Caleosin identification by mass spectrometry

The 34kDa and 29kDa protein bands were excised from Coomassie-stained gels in an EXQuest Spot Cutter (Bio-Rad). Proteins were reduced by 10mM DTT at 56°C for 45min and then alkylated by 55mM iodoacetamide at room temperature for 30min. Proteins were then subjected to in-gel digestion with trypsin (Trypsin Gold, Promega, Madison, WI, USA) at 37°C for 4h in a Digest Pro MS station (Intavis, Germany). The resulting peptides were purified using a ZipTip μ-C18 column (Millipore, Bedford, MA, USA), and eluted with 0.2% (v/v) trifluoroacetic acid (TFA). Samples (1μl) were co-crystallized with a matrix solution of 2.5mg ml−1 α-cyano-4-hydroxicinamic acid (1μl) onto MALDI (matrix-assisted laser desorption/ionization) chips. Finally, samples were subjected to MALDI-time of flight (TOF) analysis in a Voyager DE-PRO spectrometer (Applied Biosystems, Foster City, CA, USA) in positive reflection mode. An internal calibration was performed using the m/z of peptides resulting from the autolysis of trypsin.

The MS spectra were used to search algorithms against the NCBI and Swiss-Prot databases using a local Mascot™ (Matrix Science Ltd, UK) server. To be accepted for the identification, an error of <100ppm on the parent ion mass was tolerated. One missed cleavage per peptide was allowed, and carbamidomethylation for cysteine and oxidation for methionine were also considered.

Immunolocalization of caleosins in isolated OBs

Purified OBs were incubated in a 1.5ml tube with the anti-Clo3 Ab (diluted 1:30 in grinding medium) for 2h at room temperature, followed by incubation with an anti-rabbit IgG Ab conjugated with Alexa Fluor 488 (Molecular Probes), diluted 1:200 in grinding medium, for 1h at 37°C under gentle agitation. A few drops of an anti-fading solution of Citifluor (Sigma-Aldrich) were added and samples were observed with a Zeiss Axioplan epifluorescence microscope under blue light irradiation. Negative controls were treated as above, but the primary Ab was omitted.

Whole-cell immunodetection of caleosins

Mature and germinated pollen samples were fixed in a mixture of 4% (w/v) paraformaldehyde and 0.2% (v/v) glutaraldehyde prepared in 0.2M sodium cacodylate buffer (pH 7.2) at 4°C overnight. After two washes in cacodylate buffer, pollen tube cell walls were digested using a mixture of 1% (w/v) cellulase R10 (Serva, Germany) and 27U of pectinase (Sigma-Aldrich) per ml in 0.01M citrate buffer (pH 4.8) for 25min at 37°C.

After blocking with a solution containing 1% (w/v) BSA in phosphate-buffered saline (PBS) buffer (pH 7.2) for 1h, samples were incubated with the anti-Clo3 primary Ab [diluted 1:50 in PBS buffer, pH 7.2, containing 1% (w/v) BSA] overnight at 4°C. An anti-rabbit IgG Ab conjugated with Alexa Fluor 633 (Molecular Probes) diluted 1:200 in PBS buffer (pH 7.2) was used as the secondary Ab. Finally, samples were re-suspended in an anti-fading solution of Citifluor (Sigma-Aldrich). Just before examination, 10μl of Nile Red solution (prepared as described above) were added to each sample. Samples were then sequentially observed with a Nikon C1 confocal laser scanning microscope (Nikon, Japan) using a He–Ne (633nm) laser for Alexa Fluor 633 detection and an argon (488 nm) laser for Nile red staining visualization. Z-series images were collected and processed with the software EZ-C1 Gold version 2.10 build 240 (Nikon). Negative controls were treated as above, but the primary Ab was omitted.

Immunolocalization of caleosins by TEM

Samples were fixed overnight in ice-cold 0.1M cacodylate buffer, pH 7.2, containing 4% (v/v) paraformaldehyde and 0.2% (v/v) glutaraldehyde. They were then washed in the same buffer, dehydrated in a graded ethanol series, and embedded in Unicryl resin (BBInternational, UK). Ultrathin sections (70nm thick) were cut on an Ultracut microtome (Reichert-Jung, Germany) and mounted on 200 mesh formvar-coated nickel grids.

Blocking of non-specific binding sites was carried out by incubation of the sections in a solution containing 1% (w/v) BSA in PBS buffer, pH 7.2, for 1h. This was followed by treatment with the anti-Clo3 Ab (diluted 1:50 in blocking solution) overnight at 4°C. Samples were then rinsed with PBS buffer and incubated with an anti-rabbit IgG Ab conjugated with 15nm gold particles (BBInternational) diluted 1:100 in 0.5% (w/v) BSA in PBS buffer, pH 7.2, for 1h at room temperature. After washing, sections were stained with 5% (w/v) uranyl acetate for 30min at room temperature. Observations were carried out with a JEM-1011 transmission electron microscope (JEOL, Japan). Control reactions were carried out by omitting the primary Ab.

Results

Identification of a caleosin from olive pollen

Immunoblot experiments showed the presence of a polypeptide of ~30kDa, which was cross-recognized by the anti-Clo3 Ab (Fig. 1). Although this polypeptide was detected in all stages analysed, densitometric data showed the presence of quantitative differences. The highest levels of caleosin were observed immediately after hydration. At the early steps of pollen germination, levels slightly declined but they rapidly recovered after 2h of germination. From this stage onwards, caleosin levels progressively decreased, to almost completely disappear after 12h of in vitro germination.

Expression analysis of olive pollen caleosins. (A) Coomassie-stained gel of total proteins from olive pollen at maturity (MP), after hydration (H), and after 1, 2, 3, 6, and 12h of in vitro germination. (B) Immunoblot as in A probed with the...

(A) Anti-Clo3 Ab-immunoprecipitated proteins after SDS–PAGE and Coomassie staining. (B) Immunoblot as in A probed with the anti-Clo3 Ab. C1, control 1, anti-caleosin Ab probed with a secondary Ab; C2, control 2, anti-Clo3 Ab-immunoprecipitated...

OB behaviour during in vitro pollen germination

Neutral lipids were detected in both mature and germinating pollen grains as intensively fluorescent round-shaped structures in the cytoplasm (Fig. 3). The diameter of these OBs ranged from 0.2μm to 2.1μm. After hydration, ~1000±200 OBs per pollen grain were counted. An intense staining of the pollen wall surface was also observed (Fig. 3A). Neither the generative cell nor the vegetative nucleus showed any fluorescent labelling. OBs were also present in all the in vitro germination stages (Fig. 3B–F), but they showed significant differences with regard to number and size. Thus, in the cytoplasm of the pollen grain, the number of OBs decreased to <20±7.8 OBs after 12h of germination (Fig. 4A). In contrast, the average number of OBs significantly increased in the cytoplasm of the pollen tube during the first 3h of germination (Fig. 4A), reaching a maximum of 248±68.3 OBs per pollen tube. At later stages, the average number of OBs progressively decreased in the pollen tube cytoplasm. In parallel, the average size (i.e. diameter) of OBs progressively diminished during in vitro germination (Fig. 4B). Live-cell imaging of olive pollen tubes in combination with Nile red staining of OB-associated lipids allowed study of the dynamics of OB motility. During early steps of in vitro germination, OBs displayed intense dynamics, moving all along the pollen tube length following cytoplasmic streaming. This movement occurred in two ways, both from the pollen grain to the subapical region of the pollen tube and backwards. The growing pollen tube tip was not affected by this OB movement (Supplementary Video S1 available at JXB online). Once the pollen tubes grow, the movement of the OBs was initially slowed down by the presence of vacuoles, and finally restricted to the cytoplasmic regions distal from the pollen grain by the existence of callose plugs (Supplementary Video S2).

Nile red staining of OBs from olive pollen. (A) Numerous OBs fill up the cytoplasm of the vegetative cell of the hydrated pollen. The pollen wall is also stained, revealing its lipidic nature. (B–F) OBs are also visible in the pollen tube cytoplasm...

(A) Changes in the number of OBs within the olive pollen grain (open triangles) and pollen tube (open squares) during in vitro germination. (B) Average size of OBs in the olive pollen tube during in vitro germination. Z-series images corresponding to...

Immunolocalization of caleosins in isolated OBs

After purification, olive pollen OBs were visible as spherical structures with a diameter varying from 0.2μm to 2μm (Fig. 5A, B). Their lipidic nature was confirmed by Sudan Black B and Nile red staining. Immunolocalization experiments with anti-Clo3 Ab allowed confirmation of the presence of caleosins in these organelles (Fig. 5C, D). Caleosin labelling was found on the surface of OBs, often being visualized as ring-shaped fluorescent structures.

Purification of OBs from olive pollen and localization of caleosins. (A, B) Purified OBs stained with Sudan Black B and Nile red, respectively. (C, D) Localization of caleosins on isolated OBs using the anti-Clo3 Ab, at low (C) and high (D) magnification....

Immunolocalization of caleosins in the olive pollen during in vitro germination

In the mature pollen grain sections, caleosins were located in the cytoplasm, the exine, and on the pollen coat, but not in the intine (Supplementary Fig. S1A at JXB online). The control did not show any fluorescent labelling (Supplementary Fig. S1B).

Whole-mount OB staining and caleosin immunodetection were carried out in parallel in both hydrated and germinated pollen grains. In the hydrated pollen, the signal corresponding to caleosins and OBs co-localized in the cytoplasm of the vegetative cell and was visualized as a yellowish fluorescence (Fig. 6D). Additionally, a specific accumulation of OBs and caleosins was observed in the vicinity of one of the pollen grain apertures (Fig. 6D). During in vitro germination, caleosins were located in the boundaries of the OBs spread along the pollen tube and in the cytoplasm at the subapical region (Fig. 6H). Caleosins also located in the tonoplast of the vacuoles present in the pollen tube cytoplasm (Fig. 6L).

Staining of OBs and localization of caleosins in olive pollen tubes. (A–D) Optical sections of hydrated pollen grains. The signal corresponding to caleosins and OBs co-localized in the cytoplasm of the vegetative cell and was visualized as a yellowish...

At the ultrastructural level, immunogold labelling of caleosins in sections of mature and germinating pollen grains of olive confirmed the results obtained by confocal microscopy. In mature, non-germinated pollen grains, gold particles were mainly found in the cytoplasm of the vegetative cell, attached on the surface of OBs or in the vicinity of these organelles (Fig. 7A, B). Caleosins were also located in the exine, but not in the intine (Fig. 7A). During in vitro germination, gold particles were found in the pollen tube cytoplasm and the plasma membrane (Fig. 7C). In the subapical zone, where numerous organelles and membrane structures were present, gold particles were located attached to the surface of OBs (Fig. 7C). Moreover, some gold particles were found specifically localized in the surrounding ER membranes. In the region of the pollen tube distal from the apex, gold particles were mainly located in the tonoplast of vacuoles (Fig. 7D), which frequently coalesce. A control reaction with omission of the primary Ab did not show any significant gold labeling (Fig. 7E).

Ultrastructural localization of caleosins in hydrated and germinated olive pollen. (A) In the hydrated pollen, gold particles were located in the cytoplasm of the pollen grain and in the exine. (B) Higher magnification of boxed area in A, showing gold...

Discussion

A number of caleosins have been identified in mature seeds of several plant species such as rice, barley, sesame, sunflower, soybean, rape, and Arabidopsis (Frandsen et al., 1996; Chen et al., 1999; Næsted et al., 2000; Jolivet et al., 2004; Liu et al., 2005; Polxeitner et al., 2006). It was only recently that the first caleosin from pollen was identified in lily (Jiang et al., 2008). These authors cloned a cDNA fragment encoding a caleosin of ~27kDa, whose sequence is distinct from that of those caleosins found in seed OBs. The data presented here constitute, to the best of our knowledge, the first report regarding the identification and cellular localization of a caleosin isoform from pollen in an oleaginous crop plant. The use of an Ab to a heterologous caleosin from Arabidopsis (Clo-3 isoform) that is expressed in both vegetative and reproductive tissues (Partridge and Murphy, 2009) allowed identication of a putative caleosin with a molecular mass of ~30kDa, tightly associated with OBs and ER membranes. However, the possibility of the presence of additional caleosin isoforms in the olive pollen cannot be ruled out. Indeed, two caleosin isoforms, differing in their molecular masses and subcellular localization, co-exist in developing seeds of Brassica napus (Hernández-Pinzón et al., 2001).

Expression and localization studies have provided valuable clues about the putative function of caleosins in seeds (Chen et al., 1999; Næsted et al., 2000). The detection of caleosins on purified OBs from olive pollen is in good agreement with the idea that this is an integral membrane protein. Consequently, the olive pollen 30kDa caleosin was found tightly associated with purified OBs even after vigorous and repeated washes. Interestingly, although the 30kDa polypeptide was detected during the whole germination process, levels decreased coincidentally with the reduction in the number of OBs present in the cytoplasm of the pollen tube. This pattern was also observed in light-grown cotyledons of B. napus, in which levels of the 25kDa caleosin declined as OBs underwent mobilization (Hernández-Pinzón et al., 2001).

The data presented here also support the hypothesis that OBs move from the pollen grain towards the growing pollen tube as soon as the pollen grain begins to germinate (Rodríguez-García et al., 2003). OB mobilization in the olive pollen started after hydration, and progressed during the subsequent steps of pollen tube elongation. After 12h of in vitro germination, the population of OBs was almost completely metabolized. The function of OBs is still a matter of discussion. It has been proposed that OB-stored TAGs are converted into membrane lipids (Dorne et al., 1988). The localization of caleosins in both the intracellular membranes (e.g. tonoplast) and the plasmalemma is consistent with this idea. Moreover, olive pollen OBs are frequently surrounded by ER membranes (Alché et al., 1999a), which persist during pollen germination (M'rani-Alaoui, 2000). This ER network might facilitate the direct mobilization of the OB lipids into membrane components (Piffanelli et al., 1998).

The function of caleosins in the surface of OBs remains to be studied. Caleosins possess a single EF-hand domain that binds a single Ca2+ cation with low affinity. It is therefore possible that a caleosin forms either cis or trans dimeric associations with another caleosin present in the same or a distinct membrane or OB, leading to fusion events (Næsted et al., 2000). Accordingly, AtCLO1 caleosin participates in OB–vacuole interactions that affect breakdown of OBs and turnover of the vacuole membrane as storage vacuoles are remodelled during seed germination in Arabidopsis (Poxleitner et al., 2006). In the pollen tube of lily, the absence of glyoxysomes and the presence of OBs inside the vacuoles suggest that degradation of OBs might be carried out by vacuolar digestion (Jiang et al., 2007). Caleosins might therefore mediate OB–vacuole membrane fusion in the pollen tube. Calcium might also directly regulate caleosin activity. In a recent work, Hanano et al. (2006) showed that caleosins from Arabidopsis and rice seeds possess peroxygenase activity and that calcium is crucial for this activity. Whether pollen caleosins do possess peroxigenase activity or not is currently under investigation in our laboratory.

Vacuole formation is a common feature in growing pollen tubes (Noguchi, 1990; Mascarenhas, 1993; Derksen et al., 2002). Interestingly, the mechanism of vacuole biogenesis appears to be different from that in other plant tissues (Hicks et al., 2004). According to the model of Noguchi (1990), OBs enclosed by ER membranes become attached to electron-dense thin vesicles (EDTVs) in the pollen cytoplasm, undergoing lipolysis to disappear completely. In parallel with the consumption of OBs, the formation of protuberances in the expanding EDTVs and the pinching off of these protuberances leads to the formation of developing small vacuoles that fuse to each other and increase in size as they move towards the distal part of the pollen tube. In the olive, the presence of caleosins in the tonoplast of the vacuoles within the pollen tube supports the idea that OBs might be converted into vesicles and vacuoles. Caleosins docked in the tonoplast might mediate vacuole–vacuole fusion events, participating in the reorganization of membrane compartments in the growing pollen tube. Similarly, this mechanism might be mediated by Ca2+ ions as described above.

Apart from lipidic material (Pitfanelli et al., 1998), the pollen coat is a site for functional proteins involved in cell wall loosening, pollen hydration, and pollen–stigma communication (Murphy and Ross, 1998; Mayfield et al., 2001; Murphy, 2006). In the olive, the tapetum is involved in the synthesis and accumulation of several proteins that are present in the pollen coat, including some allergens such as Ole e 1 and profilins (Alché et al., 1999b; Morales et al., 2008). Moreover, OB-associated oleosin genes are expressed in the tapetal tissues of this species during anther development (Alché et al., 1999a). In the present work, caleosins were found for the first time in the exine. This finding suggests that caleosins from olive pollen may have both a sporophytic and a gametophytic origin. The analysis of the expression pattern of this caleosin during olive anther development showed that caleosins are also expressed in the tapetum (unpublished results), thus supporting this hypothesis. It has been proposed that these pollen coat-associated caleosins might have a role in pollen–stigma communication during pollination (Murphy, 2006), although this hypothesis needs to be tested in further investigations.

Supplementary data

Video S1.Time-lapse-generated movie showing the dynamics of OB motility during early olive pollen in vitro germination. Pollen grains were germinated in an appropriate medium for 3h and OBs were stained with Nile red. Both the transmitted light channel and the resulting fluorescence image were recorded. AVI file.

Video S2.Effect of vacuoles on OB traffic throughout the olive pollen tube cytoplasm during in vitro germination. Pollen grains were germinated in an appropriate medium for 3h and OBs were stained with Nile red. In order to generate the resulting time-lapse movie, both the transmitted light channel and the fluorescence images were recorded. AVI file.

Figure S1. Immunolocalization of caleosins in olive mature pollen sections. (A) Localization of caleosins (green fluorescence) in the cytoplasm of the pollen grain and the exine wall (Ex). The intine layer (arrowheads) and the apertures (asterisk) were devoid of labelling. (B) Control reaction performed by omitting the primary Ab showing only the exine autofluorescence (in red). Samples were observed with an Axioplan epifluorescence microscope (Carl Zeiss). Digital images were recorded with a ProgRes C3 camera (Jenoptik Laser) using the ProgRes CapturePro software (Jenoptik Laser). Bars=10μm.

Supplementary Material

Acknowledgments

We are grateful to Professor D Murphy (University of Glamorgan, Faculty of Health, Sport and Science, Division of Biology, Pontypridd, UK) for kindly providing us with the anti-Clo3 Ab to caleosin, and to Ms C Martínez-Sierra for expert technical assistance. We would like also to thank IFAPA “Venta del Liano” (Jaen, Spain) for providing olive material. This work was supported by the Junta de Andalucía (project P06-AGR-01791) and the Spanish Ministry of Science and Innovation (projects AGL2008-00517/AGR and BFU2008-00629/BFI). KZ and AZ thank the Junta de Andalucía for providing grant funding. KZ also thanks the CSIC for providing JAE-DOC grant funding.